The overall goal of this project is to define the evolutionary steps involved in a key aspect of the process of disease emergence; the transmission and subsequent spread of viruses from one host species to another. We will use comparative analyses to reveal the long-term phylogenetic patterns of different stages of viral emergence, followed by experimental studies to reveal the underlying processes which determine these patterns. We will examine two different viruses which have accomplished cross-species transmission within different animal systems, where we have both the ancestral viruses in the donor host species and the emerged virus from the recipient host species, but which differ in their degree of host-virus adaptation; the emergence of the canine parvovirus as a variant of a virus of cats, and the emergence of H3N8 influenza virus in canines following cross-species transmission from horses. These studies will therefore reveal the adaptive processes involved in viral emergence and have broad implications for understanding, and more importantly, forestalling such events. A major aim of the project is to examine the fitness of specific mutations associated with the emergence of the parvoviruses. The first part of this study will involve the comparative analysis of complete genome sequence data. We will then determine the fitness of intermediate viruses at two stages of parvovirus emergence; first between the feline and initial canine virus, and second the adaptive improvement of canine viruses within dogs, which also resulted in a renewed ability to infect feline cells. These intermediate viruses provide unique information on the evolutionary trade-offs that may be implicit in the process of cross-species transmission. These viruses will be tested for their viability, and other biological properties, in tissue culture. In a small number of cases, the viruses will also be tested for their ability to replicate in animals. As a second major aim we will examine the intra-host genetic diversity of viral sequences when the viruses are replicating in either their donor and recipient host, such as feline parvovirus in dogs (leading to a restricted thymic infection), or H3N8 equine influenza virus in dogs. We will examine the extent and structure of sequence diversity found after growth of the virus in the different hosts, and use comparative sequence analysis determine whether the changes seen were subject to positive or negative selection pressure, or simply reflect the intrinsic mutational spectrum. Crucially, we will also analyze the extent and structure of diversity after viruses are allowed to undergo a single round of host-host transmission, which may impose additional and/or different selection pressures. As a comparison we will also conduct selection studies using cell culture, where we will measure the rate of emergence of antibody escape mutants for both the parvoviruses and influenza viruses, as well as receptor-selected mutants of the parvoviruses